Activity regulates programmed cell death of zebrafish Rohon-Beard neurons.

Programmed cell death is a normal aspect of neuronal development. Typically, twice as many neurons are generated than survive. In extreme cases, all neurons within a population disappear during embryogenesis or by early stages of postnatal development. Examples of transient neuronal populations include Cajal-Retzius cells of the cerebral cortex and Rohon-Beard cells of the spinal cord. The novel mechanisms that lead to such massive cell death have not yet been identified. We provide evidence that electrical activity regulates the cell death program of zebrafish Rohon-Beard cells. Activity was inhibited by reducing Na+ current in Rohon-Beard cells either genetically (the macho mutation) or pharmacologically (tricaine). We examined the effects of activity block on three different reporters of cell death: DNA fragmentation, cytoskeletal rearrangements and cell body loss. Both the mao mutation and pharmacological blockade of Na+ current reduced these signatures of the cell death program. Moreover, the mao mutation and pharmacological blockade of Na+ current produced similar reductions in Rohon-Beard cell death. The results indicate that electrical activity provides signals that are required for the normal elimination of Rohon-Beard cells.

Opioid receptor subtype expression defines morphologically distinct classes of hippocampal interneurons.

The inhibition of hippocampal pyramidal cells occurs via inhibitory interneurons making GABAergic synapses on distinct segments of the postsynaptic membrane. In area CA1 of the hippocampus, the activation of mu- and delta-opioid receptors inhibits these interneurons, thereby increasing the excitability of the pyramidal cells. Through the use of selective opioid agonists and biocytin-filled whole-cell electrodes, interneurons possessing somata located within stratum oriens of hippocampal slices were classified according to the location of their primary axon termination and the expression of mu- or delta-opioid receptors. Activation of these opioid receptor subtypes resulted in outward currents in the majority of interneurons, which is consistent with their inhibition. Post hoc morphological analysis revealed that those interneurons heavily innervating the pyramidal cell body layer were much more likely to express mu-opioid receptors, whereas cells with axons ramifying in the pyramidal neuron dendritic layers were more likely to express delta-opioid receptors, as defined by the generation of outward currents. This morphological segregation of interneuron projections suggests that mu receptor activation would diminish GABA release onto pyramidal neuron somata, thereby increasing their excitability and output. Conversely, inhibition of interneurons via delta receptor activation would amplify afferent signaling to pyramidal neuron dendrites by reducing GABAergic inhibition of these structures.

Opioid inhibition of hippocampal interneurons via modulation of potassium and hyperpolarization-activated cation (Ih) currents.

The actions of mu- and delta-opioid agonists (DAMGO and DPDPE, respectively) on GABAergic interneurons in stratum oriens of area CA1 of the hippocampus were examined by using whole-cell voltage-clamp recordings in brain slices. Both agonists consistently generated outward currents of similar magnitude (15-20 pA) in the majority of cells. However, under control conditions, current-voltage (I/V) relationships revealed that only a small number of these cells (3 of 77) demonstrated clear increases in membrane conductance, associated with the activation of the potassium current known as Girk. These interneurons also exhibited a slowly activating, inwardly rectifying current known as Ih on hyperpolarizing step commands. Ih was blocked by the extracellular application of cesium (3-9 mM) or ZD 7288 (10-100 microM) but was insensitive to barium (1-2 mM). In an effort to determine whether the holding current changes were attributable to the modulation of Girk and/or Ih, we used known blockers of these ion channels (barium or cesium and ZD 7288, respectively). Extracellular application of cesium (3-9 mM) or ZD 7288 (25-100 microM) blocked Ih and significantly reduced the opioid-induced outward currents by 58%. Under these conditions the opioid agonists activated a potassium current with characteristics similar to Girk. Similarly, during barium (1-2 mM) application the opioid-induced outward currents were reduced by 46%, and a clear reduction in Ih and the whole-cell conductance was revealed. These data suggest that the opioids can modulate both Ih and Girk in the same population of stratum oriens interneurons and that the modulation of these ion channels can contribute to the inhibition of interneuron activity in the hippocampus.

Cholecystokinin increases GABA release by inhibiting a resting K+ conductance in hippocampal interneurons.

Cholecystokinin (CCK) is found co-localized with the inhibitory neurotransmitter GABA in interneurons of the hippocampus. Also, CCK receptors are found in abundance in this brain region. The possibility that CCK alters interneuron activity was examined using whole-cell current- and voltage-clamp recordings from visualized interneurons in the stratum radiatum of area CA1 in rat hippocampal slices. The effect of CCK on GABA-mediated IPSCs was also determined in pyramidal neurons. The sulfated octapeptide CCK-8S increased action potential frequency or generated inward currents in the majority of interneurons. These effects of CCK persisted in the presence of tetrodotoxin and cadmium, suggesting that they were direct. Current-voltage plots revealed that CCK-8S inhibited a conductance that was linear across command potentials and reversed near the equilibrium potential for K+ ions. The K+ channel blocker tetraethylammonium (10 mM) generated inward currents similar to those initiated by CCK, and it occluded the effect of the peptide. BaCl2 (1 mM) and 4-aminopyridine (2 mM) did not alter the effect of CCK. The CCKB receptor antagonist PD-135,158 completely blocked the inward currents generated by CCK-8S. CCK also resulted in an increase in spontaneous action potential-dependent IPSC frequency, but no changes in action potential-independent miniature IPSCs or evoked IPSCs in pyramidal neurons. These results provide evidence that CCK can depolarize hippocampal interneurons through the inhibition of a resting K+ conductance, leading to increased tonic inhibition of pyramidal neurons. This action of CCK may contribute to its anticonvulsant properties, as observed in limbic seizure models.

Interactions between the neural networks for escape and swimming in goldfish.

Interactions between neural networks for different motor behaviors occur frequently in nature; however, there are few vertebrate models for studying these interactions. One potentially useful model involves the interactions between escape and swimming behaviors in fish. Fish can produce escape bends while swimming, using some of the same axial muscles for both behaviors. Here we study the interactions between escape and swimming in a paralyzed goldfish preparation in which we can activate the networks for both behaviors. Fictive swimming was elicited by electrical stimulation in the midbrain locomotor region. During the swimming, we fired a single action potential in the reticulospinal Mauthner (M) cell, which initiates the escape behavior (Zottoli, 1977). Firing the M cell overrode the swimming motor output to produce an output appropriate for escape regardless of the phase of swimming at which it was fired. The M cell also could reset the swimming rhythm dramatically in a way that led to a smooth transition from an escape bend to one side into subsequent swimming. Both the override and reset supported predictions based on previous studies of the organization of the M-cell network. They apparently allow for a well coordinated motor output when a fish must produce an escape while swimming. The potent effects of one action potential in a single, identifiable reticulospinal neuron make this an attractive model system for future studies of the cellular basis of interactions between descending pathways and spinal rhythm-generating networks.

Fictive swimming elicited by electrical stimulation of the midbrain in goldfish.

1. We developed a fictive swimming preparation of goldfish that will allow us to study the cellular basis of interactions between swimming and escape networks in fish. 2. Stimulation of the midbrain in decerebrate goldfish produced rhythmic alternating movements of the body and tail similar to swimming movements. The amplitude and frequency of the movements were dependent on stimulus strength. Larger current strengths or higher frequencies of stimulation produced larger-amplitude and/or higher-frequency movements. Tail-beat frequency increased roughly linearly with current strength over a large range, with plateaus in frequency sometimes evident at the lowest and highest stimulus strengths. 3. Electromyographic (EMG) recordings from axial muscles on opposite sides at the same rostrocaudal position showed that stimulation of the midbrain led to alternating EMG bursts, with bursts first on one side, then the other. These bursts occurred at a frequency equal to the tail-beat frequency and well below the frequency of brain stimulation. EMG bursts recorded from rostral segments preceded those recorded from caudal segments on the same side of the body. The interval between individual spikes within EMG bursts sometimes corresponded to the interval between brain stimuli. Thus, whereas the frequency of tail beats and EMG bursts was always much slower than the frequency of brain stimulation, there was evidence of individual brain stimuli in the pattern of spikes within bursts. 4. After paralyzing fish that produced rhythmic movement on midbrain stimulation, we monitored the motor output during stimulation of the midbrain by using extracellular recordings from spinal motor nerves. We characterized the motor pattern in detail to determine whether it showed the features present in the motor output of swimming fish. The fictive preparations showed all of the major features of the swimming motor pattern recorded in EMGs from freely swimming fish. 5. The motor nerves, like the EMGs produced by stimulating midbrain, showed rhythmic bursting at a much lower frequency than the brain stimulus. Bursts on opposite sides of the body alternated. The frequency of bursting ranged from 1.5 to 13.6 Hz and was dependent on stimulus strength, with higher strengths producing faster bursting. Activity in rostral segments preceded activity in caudal ones on the same side of the body. Some spikes within bursts of activity occurred at the same frequency as the brain stimulus, but individual brain stimuli were not as evident as those seen in some of the EMGs. 6. The duration of bursts of activity in a nerve was positively and linearly correlated with the time between successive bursts (cycle time).(ABSTRACT TRUNCATED AT 400 WORDS)